The enzyme fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including anandamide (1a) and oleamide (1b) (Bracey, M. H.; Hanson, M. A.; et al. Science 2002, 298, 1793-1796; Cravatt, B. F.; Giang, D. K.; et al. Nature 1996, 384, 83-87; Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2238-2242; Patricelli, M. P.; Cravatt, B. F. Vit. Hormones 2001, 62, 95-131). The central nervous system distribution of FAAH suggests that it degrades neuromodulating fatty acid amides at their sites of action and is intimately involved in their regulation (Egertova, M.; Cravatt, B. F.; et al. Neuroscience 2003, 119, 481-496). Fatty acid amide hydrolase is currently the only characterized mammalian enzyme that is in the amidase signature family bearing an unusual catalytic Ser-Ser-Lys triad (Bracey, M. H.; Hanson, M. A.; et al. Science 2002, 298, 1793-1796; Patricelli, M. P.; Cravatt, B. F. Vit. Hormones 2001, 62, 95-131; Patricelli, M. P.; Cravatt, B. F. Biochemistry 1999, 38, 14125-14130; Patricelli, M. P.; Cravatt, B. F. J. Biol. Chem. 2000, 275, 19177-19184; Patricelli, M. P.; Lovato, M. A.; et al. Biochemistry 1999, 38, 9804-9812). Recently, the crystal structure of FAAH cocrystallized with an irreversibly-bound arachidonoyl fluorophosphonate confirmed its unusual catalytic triad and provided structural details of this enzyme (Bracey, M. H.; Hanson, M. A.; et al. Science 2002, 298, 1793-1796).
Both anandamide (1a) (Dervane, W. A.; Hanus, L.; et al. Science 1992, 258, 1946-1949) and oleamide (1b) (Boger, D. L.; Henriksen, S. J.; et al. Curr. Pharm. Des. 1998, 4, 303-314; Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580-590; Cravatt, B. F.; et al. Science 1995, 268, 1506-1509) have emerged as prototypical members of the class of bioactive lipid amides (Boger, D. L.; Fecik, R. A.; et al. Bioorg. Med. Chem. Lett. 2000, 10, 2613-2616; Lang, W.; Qin, C.; et al. J. Med. Chem. 1999, 42, 896-902) that serve as chemical messengers (FIG. 1). Anandamide (1a), the most recognizable member of the endogenous fatty acid ethanolamides (Schmid, H. H. O.; Schmid, P. C.; Natarajan, V. Prog. Lipid Res. 1990, 29, 1-43), binds and activates both the central type-1 (CB1) and peripheral type-2 (CB2) cannabinoid receptors. Anandamide (1a), and members of the cannabinoid family (Lambert, D. M.; Fowler, C. J. J. Med. Chem. 2005, 48, 5059-5087), have been implicated in the modulation of nociception (Calignano, A.; La Rana, G.; et al. Nature 1998, 394, 277-281; Cravatt, B. F.; Lichtman, A. H. J. Neurobiol. 2004, 61, 149-160; Walker, J. M.; Huang, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12198-12203), feeding (Gomez, R.; Navarro, M.; et al. J. Neurosci. 2002, 22, 9612-9617; Williams, C. M.; Kirkham, T. C. Physiol. Behav. 2002, 76, 241-250), emesis, anxiety (Kathuria, S.; Gaetani, S.; et al. Nat. Med. 2003, 9, 76-81), cell proliferation (Melck, D.; Rueda, D.; et al. FEBS Lett. 1999, 463, 235-240; Yamaji, K.; Sarker, K. P.; et al. Thromb. Haemostasis 2003, 89, 875-884), inflammation (Massa, F.; Marsicano, G.; et al. J. Clin. Invest. 2004, 113, 1202-1209), memory (Mallet, P. E.; Beninger, R. J. Psychopharmacology 1998, 140, 11-19) and neuroprotection after brain injury (Panikashvili, D.; Simeonidou, C.; et al. Nature 2001, 413, 527-531). Thus, the cannabinoids have clinical relevance for analgesia, anxiety, epilepsy, cachexia, cancer, Alzheimer's disease as well as other neurodegenerative diseases (Axelrod, J.; Felder, C. C. Neurochem. Res. 1998, 23, 575-581; Di Marzo, V.; Bisogno, T.; et al. Curr. Med. Chem. 1999, 6, 721-744; Martin, B. R.; Mechoulam, R.; et al. Life Sci. 1999, 65, 573-595).
Oleamide (1b) was found to accumulate in the cerebrospinal fluid of animals under conditions of sleep deprivation and to induce physiological sleep in a dose dependent manner (Boger, D. L.; Henriksen, S. J.; et al. Curr. Pharm. Des. 1998, 4, 303-314; Cravatt, B. F.; et al. Science 1995, 268, 1506-1509). It modulates serotonergic systems (Cheer, J. F.; Cadogan, A.-K.; et al. Neuropharmacology 1999, 38, 533-541; Thomas, E. A.; Cravatt, B. F.; et al. J. Neurochem. 1999, 72, 2370-2378; Boger, D. L.; Patterson, J. E.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4102-4107) and GABAergic transmission (Lees, G.; Dougalis, A. Brain Res. 2004, 997, 1-14; Yost, C. S.; Hampson, A. J.; et al. Anesth. Analg. 1998, 86, 1294-1299), decreases body temperature and locomotor activity (Huitrón-Reséndiz, S.; Gombart, L.; et al. Exp. Neurol. 2001, 172, 235-243), and blocks glial gap junction cell-cell communication (Boger, D. L.; Patterson, J. E.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4810-4815; Guan, X.; Cravatt, B. F.; et al. J. Cell Biol. 1997, 139, 1785-1792). The dual inhibition of presynaptic Na+ channels and postsynaptic GABAA receptors suggests oleamide (1b) may possess a mode of action common to drugs that are widely used for the treatment of anxiety, sleep disorders, and epilepsy and that it represents an endogenous ligand for such depressant drug sites in the mammalian brain. Oleamide (1b) decreases body temperature and locomotor activity (Huitrón-Reséndiz, S.; Gombart, L.; et al. Exp. Neurol. 2001, 172, 235-243), and exhibits the characteristic in vivo analgesic and cannabinoid behavioral effects of anandamide (Cheer, J. F.; Cadogan, A.-K.; et al. Neuropharmacology 1999, 38, 533-541; Mechoulam, R.; Fride, E.; et al. Nature 1997, 389, 25-26), albeit without apparent cannabinoid receptor activation. It has been suggested that the cannabinoid behavioral effects of oleamide (1b) may be mediated through an as yet unknown distinct pharmacological target (Lees, G.; Dougalis, A. Brain Res. 2004, 997, 1-14). Because oleamide (1b) may play an important role in sleep, it may provide opportunities for the development of sleep aids that induce physiological sleep lacking the side effects of the sedative-hypnotics (e.g., benzodiazepene class), which include rebound insomnia, anterograde amnesia and suicide abuse potential.
The pharmacological actions of anandamide (1a) and oleamide (1b) are terminated by FAAH (FIG. 1) (Bracey, M. H.; Hanson, M. A.; et al. Science 2002, 298, 1793-1796; Cravatt, B. F.; Giang, D. K.; et al. Nature 1996, 384, 83-87; Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2238-2242; Patricelli, M. P.; Cravatt, B. F. Vit. Hormones 2001, 62, 95-131). Studies with FAAH knockout mice have not only shown that FAAH is a key regulator of fatty acid amide signaling in vivo, but that the animals exhibit a significantly augmented behavioral response (e.g., increased analgesia, hypomotility, catalepsy) to administered anandamide (1a) and oleamide (1b), that correlated with a CB1-dependent analgesic phenotype (Clement, A. B.; Hawkins, E. G.; et al. J. Neurosci. 2003, 23, 3916-3923; Cravatt, B. F.; Demarest, K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9371-9376; Cravatt, B. F.; Saghatelian, A.; et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10821-10826; Lichtman, A. H.; Shelton, C. C.; et al. Pain 2004, 109, 319-327). As such, FAAH has emerged as an interesting new therapeutic target for a range of clinical disorders (Lambert, D. M.; Fowler, C. J. J. Med. Chem. 2005, 48, 5059-5087; Cravatt, B. F.; Lichtman, A. H. Curr. Opin. Chem. Biol. 2003, 7, 469-475).
Due to the potentially exciting therapeutic potential of inhibiting FAAH, there has been increasing interest in the development of potent inhibitors (FIG. 2) (Kathuria, S.; Gaetani, S.; et al. Nat. Med. 2003, 9, 76-81; Boger, D. L.; Miyauchi, H.; et al. J. Med. Chem. 2005, 48, 1849-1856; Boger, D. L.; Sato, H.; et al. Bioorg. Med. Chem. Lett. 1999, 9, 265-270; Boger, D. L.; Sato, H.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5044-5049; De Petrocellis, L.; Melck, D.; et al. Biochem. Biophys. Res. Commun. 1997, 231, 82-88; Deutsch, D. G.; Omeir, R.; et al. Biochem. Pharmacol. 1997, 53, 255-260; Deutsch, D. G.; Lin, S.; et al. Biochem. Biophys. Res. Commun. 1997, 231, 217-221; Du, W.; Hardouin, C.; et al. Bioorg. Med. Chem. Lett. 2005, 15, 103-106; Edgemond, W. S.; Greenberg, M. J.; et al. J. Pharmacol. Exp. Ther. 1998, 286, 184-190; Fernando, S. R.; Pertwee, R. G. Br. J. Pharmacol. 1997, 121, 1716-1720; Koutek, B.; Prestwich, G. D.; et al. J. Biol. Chem. 1994, 269, 22937-22940; Patricelli, M. P.; Patterson, J. P.; et al. Bioorg. Med. Chem. Lett. 1998, 8, 613-618; Patterson, J. E.; Ollmann, I. R.; et al. J. Am. Chem. Soc. 1996, 1996, 5938-5945; Tarzia, G.; Duranti, A.; et al. ChemMedChem 2006, 1, 130-139; Tarzia, G.; Duranti, A.; et al. J. Med. Chem. 2003, 46, 2352-2360; Mor, M.; Rivara, S.; et al. J. Med. Chem. 2004, 47, 4998-5008; Muccioli, G. G.; Fazio, N.; et al. J. Med. Chem. 2006, 49, 417-425). These include the discovery that the endogenous sleep-inducing molecule 2-octyl γ-bromoacetoacetate is an effective FAAH inhibitor (Patricelli, M. P.; Patterson, J. P.; et al. Bioorg. Med. Chem. Lett. 1998, 8, 613-618), a series of reversible inhibitors bearing an electrophilic ketone (Boger, D. L.; Sato, H.; et al. Bioorg. Med. Chem. Lett. 1999, 9, 265-270; Koutek, B.; Prestwich, G. D.; et al. J. Biol. Chem. 1994, 269, 22937-22940; Patterson, J. E.; Ollmann, I. R.; et al. J. Am. Chem. Soc. 1996, 1996, 5938-5945) (e.g., trifluoromethyl ketone-based) that have not proven selective for FAAH over other mammalian serine hydrolases (Leung, D.; Du, W.; et al. Bioorg. Med. Chem. Lett. 2005, 15, 1423-1428) and a set of irreversible inhibitors (De Petrocellis, L.; Melck, D.; et al. Biochem. Biophys. Res. Commun. 1997, 231, 82-88; Deutsch, D. G.; Omeir, R.; et al. Biochem. Pharmacol. 1997, 53, 255-260; Deutsch, D. G.; Lin, S.; et al. Biochem. Biophys. Res. Commun. 1997, 231, 217-221; Edgemond, W. S.; Greenberg, M. J.; et al. J. Pharmacol. Exp. Ther. 1998, 286, 184-190; Fernando, S. R.; Pertwee, R. G. Br. J. Pharmacol. 1997, 121, 1716-1720) (e.g., fluorophosphonates and sulphonyl fluorides). Recently, two classes of inhibitors have been disclosed that provide significant opportunities for the development of an inhibitor with therapeutic potential. One class is the aryl carbamates (e.g., URB-597 2a; FIG. 2) that acylate an active site catalytic serine and which were shown to exhibit anxiolytic activity and induce analgesia (Kathuria, S.; Gaetani, S.; et al. Nat. Med. 2003, 9, 76-81; Tarzia, G.; Duranti, A.; et al. ChemMedChem 2006, 1, 130-139; Tarzia, G.; Duranti, A.; et al. J. Med. Chem. 2003, 46, 2352-2360; Mor, M.; Rivara, S.; et al. J. Med. Chem. 2004, 47, 4998-5008; Hohmann, A. G.; Suplita, R. L.; et al. Nature 2005, 435, 1108-1112). However, the selectivity of such aryl carbamate inhibitors is low and recent studies illustrate that either no or minimal selectivity is achieved (e.g., other targets of URB-597 2a are carboxylesterase 6 and triacylglyceride hydrolase) (Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179-1187; Lichtman, A. H.; Leung, D.; et al. J. Pharmacol. Exp. Ther. 2004, 311, 441-448; Alexander, J. P.; Cravatt, B. F. J. Am. Chem. Soc. 2006, 128, 9699-9704). A second class is the α-ketoheterocycle-based inhibitors of which some are extraordinarily potent (e.g., OL-135 2b; FIG. 2) (Boger, D. L.; Miyauchi, H.; et al. J. Med. Chem. 2005, 48, 1849-1856; Boger, D. L.; Sato, H.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5044-5049; Du, W.; Hardouin, C.; et al. Bioorg. Med. Chem. Lett. 2005, 15, 103-106; Leung, D.; Du, W.; et al. Bioorg. Med. Chem. Lett. 2005, 15, 1423-1428; Boger, D. L.; Miyauchi, H.; et al. Bioorg. Med. Chem. Lett. 2001, 11, 1517-1520). These competitive inhibitors bind to FAAH via reversible hemiketal formation with an active site serine, and are not only potent and extraordinarily selective for FAAH versus other mammalian serine hydrolases (Boger, D. L.; Miyauchi, H.; et al. J. Med. Chem. 2005, 48, 1849-1856; Leung, D.; Du, W.; et al. Bioorg. Med. Chem. Lett. 2005, 15, 1423-1428), but many are efficacious in vivo and promote analgesia (Lichtman, A. H.; Leung, D.; et al. J. Pharmacol. Exp. Ther. 2004, 311, 441-448; Chang, L.; Luo, L.; et al. Br. J. Pharmacol. 2006, 148, 102-113).
The present invention concerns the synthesis and evaluation of a systematic series of α-keto oxazole inhibitors having variations at the C2 acyl side chain along with results of the proteome-wide selectivity screening of the candidate inhibitors. The screening protocol is described by Leung, D.; Hardouin, C.; et al. Nature Biotech. 2003, 21, 687-691.